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The present invention relates to methods for diagnosing or assessing an individual""s susceptibility to a neurological disorder or a neuronal injury. The invention also relates to therapeutic methods for treating an individual suffering from a neurological disorder or a neuronal injury and methods for identifying agents that can be administered to treat such an individual.
Uncoupling proteins (UCPs; thermogenins) are proton-translocating proteins located in the inner mitochondrial membrane that play a role in metabolic processes, particularly non-shivering thermogenesis. The first UCP (UCP-1) was found to be localized in the brown adipose tissue, specialized fat cells that function in heat generation and energy balance. Hibernating and cold-adapted animals have significant stores of such tissue. The evidence indicates that UCPs function to maintain the core body temperature of hibernating mammals and other cold-adapted animals by raising the resting metabolic rate of the animals (see, e.g., Nicolls, D. G. and Locke, R. M. (1984) Physiol. Rev. 64:2-40; and Rothwell, N.J., and Stock, M. J. (1979) Nature 281:31-35).
As the name indicates, UCPs serve an uncoupling function, specifically by uncoupling proton flux through the mitochondrial membranes and ATP synthesis. The mitochondrial oxidation of metabolites (e.g., pyravate and fatty acids) is accompanied by proton transport out of the mitochondrial matrix, thereby generating a transmembrane proton gradient. The protons re-enter the mitochondria through the protein ATP synthase and drive the synthesis of ATP. The UCPs, however, provide a route for the re-entry of the protons that is uncoupled to ATP synthesis. Consequently, instead of the proton gradient resulting in the generation of ATP, UCPs act to covert the proton gradient into heat energy and increase the rate of respiration. Exposure to the cold triggers the neural and hormonal stimulation of brown adipose tissue, which in turn increases UCP-mediated proton transport and heat production (see, e.g., Susulic, V. S., and Lowell, B. B. (1996) Curr. Opin. in Endocrinol. and Meta. 3:44-50). Studies conducted with various transgenic models have demonstrated that a reduction in UCP activity correlates with the development of obesity and diabetes (see, e.g., Lowell, B. B., et al. (1993) Nature 366:740; and Kopecky, J. et al. (1995) J. Clin. Invest. 96:2914-23).
While humans have a UCP-1 gene that is active in brown fat, these fat deposits disappear shortly after birth (see, e.g., Bouillaud, et al. (1985) Proc. Natl. Acad. Sci. 82:445-448). Nonetheless, measurements showing that 25% to 30% of the oxygen that humans and other animals utilize to metabolize their food is used to compensate for mitochondrial proton leaks suggested the presence of other UCPs in humans. In fact, several human UCPs have now been identified.
One such UCP is referred to in the literature as UCP-2 or UCPH. The gene encoding this protein maps to human chromosome 11 and has been linked to hyperinsulinemia and obesity. UCP-2 is reported to be expressed in various adult tissue, including brain, muscle and fat cells (see, e.g., Fleury, et al. (1997) Nat. Genet. 15:269-272; Tartaglia, et al. PCT Publication No. WO 96/05861; Gimeno, et al. (1997) Diabetes 46:900-906; and Boss, et al. (1997) FEBS Letters 408:39-42). Allelic variants of UCP-2 appear to have been identified. While some UCP-2 proteins have an alanine at position 55 (see, Fleury, supra, and PCT Publication No. WO 00/06087), other UCP-2 proteins have a valine (see, PCT Publication WO 96/05861). At position 219, some UCP-2 proteins have a threonine (see, PCT Publication WO 96/05861 and PCT Publication WO 00/06087), whereas other UCP-2 proteins have an isoleucine (see, Fleury, supra). Methods for screening for allelic variants are discussed in PCT Publication WO 99/48905.
A third human UCP (UCP-3) has also been recently reported. This UCP is preferentially expressed in human skeletal muscle. The gene encoding this particular UCP maps to human chromosome 11, adjacent to the gene for UCP-2. Studies indicate that UCP-3 expression can be regulated by known thermogenic stimuli such as leptin, xcex2-adrenergic agonists and thyroid hormone (see, e.g., PCT publication WO 98/45313; Boss, et al., (1997) FEBS Letters 408:39-42; Vidal-Puig, et al. (1997) J. Biol. Chem. 272:24129-24132; Solanes et al. (1997) J. Biol. Chem. 272:25433-25436; and Gong, et al. (1997) J. Biol. Chem. 272:24129-24312).
A fourth human UCP (UCP-4) has been identified. This UCP is expressed in a number of different tissues including, brain, heart, pancreas and muscle tissue (see, e.g., PCT Publication WO 00/04037). Another human UCP (UCP5/BMCP1) is most abundantly expressed in the brain, and at lower levels in most peripheral organs (Sanchis, et al. (1998) J. Biol. Chem. 273: 36411, and PCT Publication WO 00/032624).
Because of the role UCPs play in uncoupling the oxidation of metabolites and the storage of the resulting energy in the form of ATP, UCPs have been viewed primarily as targets for controlling a number of weight disorders (e.g., obesity and underweight disorders), as well as related diseases (e.g., diabetes). However, there is a paucity of information regarding other physiological functions of UCP and how UCP can be utilized in other types of applications other than weight-related applications.
Provided herein are various methods for diagnosing and treating various neurological disorders and neuronal injuries, particularly stroke and ischemic stroke. Methods for screening agents to identify agents useful in treating neurological disorders and injuries are also provided.
More specifically, certain methods involve diagnosing the occurrence of a stroke or assessing a patient""s susceptibility to a stroke by detecting in a patient sample an elevated level of UCP-2 expression. In some methods, detection is accomplished by detecting elevated levels of UCP-2 transcript. Other methods involve detecting an elevated level of UCP-2 polypeptide. Elevated levels of UCP-2 polypeptide can be detected using various immunological techniques such as ELISA assays.
Some of the diagnostic methods provided herein involve assessing a patient""s risk of having a stroke. Such methods involve comparing the level of UCP-2 expression in a test sample from the patient with a baseline value, wherein an elevated level of UCP-2 expression in the patient sample relative to the baseline indicates that the patient is at risk for stroke. A variety of baseline levels can be utilized in these methods. In some instances the baseline is the level of UCP-2 expression in a patient sample obtained previously. In other methods, the baseline value is an average value, a mean value or another statistical value for a population of control individuals.
Certain treatment methods provided herein involve treating a subject having or being susceptible to a neurological disorder or a neuronal injury by administering to the subject an effective amount of an agent that increases the activity of UCP-2. The neurological disorders or neuronal injuries that are amenable to the methods include stroke, Parkinson""s disease, Huntington""s disease, inherited ataxias, motor neuron diseases, Alzheimer""s disease, epilepsy and traumatic brain injury. If the subject is susceptible to the neurological disorder or the neuronal injury, the subject is administered a prophylactic amount of the agent prior to occurring of the disorder or the injury. If, however, the subject has already suffered the neurological disorder or the neuronal injury, then the subject is administered a therapeutic amount of the agent. The agent which increases the activity of UCP-2 can be co-administered with various other agents, including, for example, agents that increase permeability of the blood/brain barrier and/or blood anticoagulants. In certain treatment methods, the agent is a purified UCP-2 polypeptide administered with a pharmaceutically acceptable carrier.
Certain treatment methods involve administering agents that stimulate the synthesis or expression of UCP-2 or a UCP-2 inducing agent. In some methods, the agent administered is a nucleic acid that encodes UCP-2 or a UCP-2 inducer. In such instances, the nucleic acid can be inserted into a viral vector or other expression vectors. The viral vector can also include a promoter operably linked to the nucleic acid which selectively drives expression in nerve cells. The promoter can be a UCP-2 promoter or a heterologous promoter. In certain methods, the viral vector is introduced into the cerebrospinal fluid; in other methods, the vector is injected into the intraventricular space. Still other treatment methods also involve producing ex vivo genetically-modified neuronal or non-neuronal stem cells that harbor the vector that includes a nucleic acid encoding UCP-2. The modified stem cells are then introduced into the intracerebroventricular space or into the cerebrospinal fluid.
A variety of screening methods is provided. Certain of these methods involve screening for an agent useful for treating a neuronal injury (e.g., stroke, traumatic brain injury) or a neurological disorder (e.g., Parkinson""s disease, Alzheimer""s disease, or epilepsy) by identifying an agent that upregulates UCP-2 expression and/or activity. Some of the screening methods involve: (a) administering to a test subject a test compound, wherein the test subject is a mammal other than a human; (b) preconditioning the test subject; and (c) determining in a sample from the test subject the expression level of UCP-2 to identify a test agent that upregulates UCP-2 expression in the test subject.
In other screening methods, agents useful for treating a neurological disorder or a neuronal injury are identified by identifying an agent that inhibits cellular apoptosis. Often such methods are conducted to identify agents useful in treating stroke or ischemic stroke. Certain screens identify compounds that inhibit the loss of mitochondrial membrane potential. Other screens provided herein identify agents that inhibit opening of the mitochondrial transition pore and release of cytochrome c from mitochondria and/or agents that inhibit the activation of caspases, as these events are associated with cellular apoptosis.